Low oxygen content compositions of 1alpha, 25-dihydroxycholecalciferol

ABSTRACT

The invention relates to stable compositions comprising 1α, 25-dihydroxycholecalciferol, a unit dose system comprising the same in a sealed vessel, and a process for preparing them.

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/365,532 filed Aug. 2, 1999.

TECHNICAL FIELD OF INVENTION

[0002] The invention relates to pharmaceutical compositions comprising1α, 25-dihydroxycholecalciferol, a unit dose system comprising the samein a sealed vessel, and a process for preparing them.

BACKGROUND OF THE INVENTION

[0003] The compound 1α, 25-dihydroxycholecalciferol is the activecomponent in a variety of pharmaceutical products having use intreatment of diseases related to abnormal serum calcium level. Thecompound has the generic name calcitriol, and stimulates intestinalabsorption of calcium and phosphorous, making it a beneficialconstituent of replacement therapy and treatment of hypocalcemia inpatients suffering from a diminished ability to produce or metabolizevitamin D.

[0004] 1α, 25-dihydroxycholecalciferol is reported in the literature todemonstrate an improved effect on conditions in both children andadults. Use of 1α, 25-dihydroxycholecalciferol includes successfultreatment of renal osteodystrophy, hypoparathyroidism, osteomalacia,osteoporosis, hepatic osteodystrophy, vitamin D-resistant rickets,vitamin D-dependent rickets, childhood renal failure and neonatalhypocalcemia. The compound is most widely used in patients with chronicrenal failure.

[0005] The compound 1α, 25-dihydroxycholecalciferol is known to besensitive to the presence of oxygen in the form of free molecules andother reactive forms. Sensitivity of the drug manifests itself in theform of producing oxidation-based degradants in solution, resulting inthe reduction of potency and a change in solution color. The negativeeffects tend to manifest themselves even after terminal sterilizationand during the shelf life of the product.

[0006] To decrease overall oxygen sensitivity of the drug solution, mostcompositions of 1α, 25-dihydroxycholecalciferol generally employ the useof buffers andlor chelating agents to maintain drug stability asmeasured by potency and color. See, U.S. Pat. Nos. 4,308,264; 4,948,788;and 5,182,274. The buffer systems and/or chelating agents in suchproducts allow the 1α, 25-dihydroxycholecalciferol solutions to beprepared under less stringent conditions for manufacturing and storage.

[0007] However, it has been described that the use of buffers and/orchelating agents is related to certain levels of aluminum in the drugsolution. Recently, the Food and Drug Administration has recognized thatcertain levels of aluminum can be undesirable in pharmaceuticalcompositions. Aluminum in Large and Small Volume Parenterals Used inTotal Parenteral Nutrition, Federal Register, FDA, HHS Action: proposedrule 21 CFR Part 201, Jan. 5, 1998. As a result, it is desirable toprovide compositions having the lowest levels of aluminum that can beattained. To obtain a low aluminum composition of 1α,25-dihydroxycholecalciferol, buffers and chelating agents have beenremoved.

[0008] Compositions of 1α, 25-dihydroxycholecalciferol which do notemploy the use of buffers or chelating agents are not widely known.Literature recognizing any oxygen stable compositions of such nature hasnot been described. Pending U.S. application Ser. No. 08/900,981 in thename of Li et al. describes 1α, 25-dihydroxycholecalciferol compositionshaving low levels of aluminum in solution, which can be achieved in partby removing the buffer and chelating components from the solution.Japanese patent 05-238,936 discloses an aqueous formulation ofcalcitriol comprising a nonionic surfactant and ascorbic acid. Standardmethods of removal or preventing the introduction of oxygen aredescribed, but there is no appreciation of the stringent controls onoxygen limits suitable for preparing a stable low aluminum formulation.

[0009] We have now determined that the removal of the buffers andchelating agents from the desired low aluminum formulations diminishesthe ability of the composition to compensate for the oxygen-sensitivityof the 1α, 25-dihydroxycholecalciferol active agent and otheroxygen-sensitive components of the composition. The presence of oxygenin the 1α, 25-dihydroxycholecalciferol in aqueous formulationpredominantly comes from exposure to oxygen in the unfilled area of acontainer carrying the drug solution (headspace) as well as theabsorption of gaseous oxygen into the drug solution.

[0010] Commercially available methods for controlling oxygen can achievean oxygen content level of 3-10% in the headspace. These methods forcontrolling the oxygen content of the headspace are insufficient forreliably controlling the levels of oxygen necessary to obtain a stablelow aluminum formulation of 1α, 25-dihydroxychole-calciferol havingdesirable potency and color over the shelf life of the composition.

[0011] Stringent control of the oxygen levels in the headspace providebeneficial effects for decreasing the total oxygen content in theoverall composition, including the container headspace and the dissolvedoxygen that is absorbed into the drug solution. Consequently, purging asufficient amount of oxygen from headspace of the composition affords anoverall more stable formulation of the oxygen-sensitive drug. We havedetermined that to provide a stable composition of a 1α,25-dihydroxycholecalciferol formulation, which is essentially free ofbuffer or chelating agent, the composition should have less than orequal to about 2.0% oxygen by volume when determined relative to thevolume of the headspace in the container.

[0012] Therefore, one object of the present invention relates toproviding a composition of 1α, 25-dihydroxycholecalciferol having lowoxygen levels of less than or equal to 2.0% oxygen in the headspace of acontainer. These oxygen levels are suitable to provide a stable lowaluminum formulation having less than or equal to about 1 part permillion (ppm) of aluminum that is essentially free of buffer orchelating agent.

[0013] Another object of the invention relates to preparing a unit dosesystem in a sealed vessel comprising an aqueous solution of atherapeutic amount of 1α, 25-dihydroxycalciferol having oxygen levelsless than or equal to 2.0% oxygen in the headspace.

[0014] Yet another object of the invention relates to a process forpreparing compositions of 1α, 25-dihydroxycholecalciferol having anamount of less than or equal to 2.0% oxygen in the headspace of acontainer. Preferably, the container is a unit dose vial.

SUMMARY OF THE INVENTION

[0015] In one aspect, the invention relates to a composition comprisinga therapeutically effective amount of 1α, 25-dihydroxycholecalciferol inan aqueous solution having less than or equal to 2.0% oxygen in theheadspace of a container when determined immediately after containersealing, said solution consisting essentially of a solubilizing agentand an antioxidant. The compositions provide a stable low aluminumformulation having less than or equal to about 1 ppm of aluminum duringthe shelf life of the composition. The composition is essentially freeof buffer and/or chelating agents.

[0016] In another aspect, the invention relates to a unit dose systemcomprising an aqueous solution of a therapeutically effective amount of1α, 25-dihydroxycholecalciferol in a sealed vessel having an amount ofoxygen less than or equal to 2.0% oxygen in the headspace whendetermined immediately after sealing, said solution of 1α,25-dihydroxycholecalciferol consists essentially of a solubilizing agentand an antioxidant.

[0017] Yet another aspect of the invention relates to a process forpreparing a low aluminum composition of aqueous 1α,25-dihydroxycholecalciferol, comprising the steps of:

[0018] a. flushing an empty container in the presence of an inert gas,

[0019] b. maintaining an inert gas environment by filling an aqueoussolution of 1α, 25-dihydroxycholecalciferol into said container whileconsistently flushing with an inert gas,

[0020] c. suitably minimizing the re-introduction of oxygen during steps(a) and (b) above and during transport of the container, and

[0021] d. sealing said container in a manner to minimize the presence ofoxygen in the container headspace.

[0022] The composition can be prepared in a vial according to steps (a)through (c) as described above; placing an appropriate means of closureinto the opening of the filled container in a manner which allows theexchange of gas from an interior side of the container to an exteriorside of the container; vacuum treating the container headspace andpurging the container headspace with an inert gas; and repeating thevacuum treating and purging step to control oxygen content in thecontainer headspace before suitably sealing the container.

[0023] In an ampoule, the process is carried out according to steps (a)through (d), wherein the manner of sealing the container is known in theart, typically by flame-sealing.

[0024] Still yet another aspect of the invention relates to a productmade by the process as described above. The product can be characterizedby having a therapeutically effective amount of 1α,25-dihydroxycholecalciferol in an aqueous solution having less than orequal to 2.0% oxygen in the headspace when determined immediately aftercontainer sealing, said solution consisting essentially of asolubilizing agent and an antioxidant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a schematic representation of an apparatus suitable foraccomplishing preliminary preparation of a suitable container for thecomposition of the invention, which is described herein and furtherconnoted as Preliminary Processing Stage (A).

[0026]FIG. 2 is a schematic representation of an apparatus suitable forfilling suitable container to prepare a composition of the invention,which is described herein and further connoted as the Filling Stage (B).

[0027]FIG. 3 is a schematic representation of a means of flushing asuitable container to prepare a composition of the invention.

[0028]FIG. 4 is a schematic representation of a preferred means forattaching the filling means to prepare a composition of the invention.

[0029]FIG. 5 is a schematic representation of an apparatus suitable forplacing a means of closure in a vented position on a container thatreceives the composition of the invention, which is described herein andfurther connoted as the Filling Stage (C).

[0030]FIG. 6 is a schematic representation of an apparatus suitable forcollecting a plurality of containers containing the composition of theinvention, which is described herein and further connoted as theAccumulation Stage (D).

[0031]FIG. 7 is a schematic representation of a chamber suitable fordrawing a vacuum and purging oxygen from the composition of theinvention, which is described herein and further connoted as the Vacuumand Purging Process Stage (E).

[0032]FIG. 8 is a schematic representation of an apparatus suitable forcontrolling a vacuum and purging oxygen from the composition of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

[0033] 1. Definitions

[0034] As used herein the term “headspace” refers to an unfilled area ina container carrying drug solution. The container is typically sealed toprevent exchange of any gasses present above the filled solution portionof the container with the external gaseous environment outside of thesealed container.

[0035] As used herein the term “immediately after container sealing”refers to the period of time before the terminal sterilization step, ifa product is terminally sterilized, and after the sealing of thecontainer. In a product that is aseptically filled, the term refers to areasonable amount of time after the final sealing of the container.

[0036] As used herein the term “unit dose system” refers to apharmaceutical dosage form suitable for a single dose to a patient inneed of treatment. Generally, a unit dose system refers to a suitablecontainer, such as a vial or an ampoule, containing a therapeuticallyeffective formulation and a suitable amount of inert gas in thecontainer. One common unit dose system is a unit dose vial. Anothercommon unit dose system is the unit dose ampoule.

[0037] As used herein the term “during the shelf life of the product”refers to any point in time during the pharmaceutically accepted termfor storing a finished pharmaceutical product. For a 1α,25-dihydroxycholecalciferol product, the typical shelf life is fromabout 12 months to about 18 months.

[0038] 2. Compositions

[0039] A composition of the invention will provide an aqueousformulation of 1α, 25-dihydroxycholecalciferol demonstrating improvedstability due to controlled levels of oxygen in an amount less than orequal to 2.0% in the headspace of a container. The composition issuitable for parenteral administration. The intravenous route ofadministration is preferred.

[0040] The active agent is 1α, 25-dihydroxycholecalciferol, which isrepresented by the structure below:

[0041] The compound has a molecular formula of C₂₇H₄₄O₃, having amolecular weight of about 416.65. The compound is soluble in organicsolvents and is practically insoluble in water. Preferably, the activeagent comprises from about one to about two micrograms for eachmilliliter of aqueous solution.

[0042] Suitable solubilizing agents for the formulations of the presentinvention are nonionic surfactants which generally comprise thepolyoxalkylene compounds, alcohols, and polysorbate compounds. It isunderstood by those skilled in the art that the solubilizing agents usedin the formulations of the present invention should be of apharmaceutically acceptable grade and acceptable for administration intohumans.

[0043] Preferably, the solubilizing agents are selected fromdimethylacetamide, polyethylene glycol 400 (PEG 400), polyethyleneglyclol 200 (PEG 200), ethanol, isopropanol, 1,3-butanediol, propyleneglycol, dimethylsulfoxide, glycerin, polysorbate 20, polysorbate 40,polysorbate 60, and polysorbate 80. In particular, compounds such aspolyoxytheylenesorbitan monolaureate and analogous compounds work welldue to their solubilizing effectiveness and low toxicity. Otherpolyoxyethylene ester solubilizing agents which can be used includethose listed in, but not intended to be limited to, the 1999 Sigma®Chemical Company catalog. The most preferred solubilizing agent ispolysorbate 20.

[0044] Generally, the solubilizing agent is used to facilitate thedissolution of the 1α, 25-dihydroxycholecalciferol into solution.Concentrations of the solubilizing agent should be pharmaceuticallyacceptable and suitable for facilitating the active agent into solution.The suitable amount of solubilizing agent used will depend on theparticular agent used in the formulation. It is preferred that theminimum amount of solubilizing agent suitable for dissolving the activeagent is used in the composition. Preferably, the polysorbate 20solubilizing agent comprises at least 0.32% by weight relative to theweight of the drug solution (wt./wt.).

[0045] Antioxidants are used to help prevent oxidation of the drug inthe product. In the formulations of the present invention, theantioxidant is quickly oxidized thereby minimizing oxidation of 1α,25-dihydroxycholecalciferol. Exemplary antioxidants include, but are notlimited to, water soluble antioxidants. A suitable amount of antioxidantwill allow for about 10% to about 20% wt./wt. of the original amount ofantioxidant to remain in the composition at the end of shelf lifewithout being consumed. Preferably, the composition comprises from about0.001% wt./wt. to about 1.5% wt./wt. of the antioxidant, depending onthe nature of the antioxidant. Representative antioxidants suitable forthe invention are described in Table 1 and are accompanied by apreferred range for each antioxidant. TABLE 1 ascorbic acid 0.01-0.5%metal ascorbates  0.2-1.0% sodium bisulfite 0.05-1.0% sodium 0.025-0.2% metabisulfite ascorbyl palmitate 0.01-1.0% sodium sulfite 0.01-1.0%sodium formaldehyde 0.005-0.15% acetone sodium 0.2%  sulfoxylatebisulfite tocopherol 0.05-0.5% dilaurylthiodi- 0.01-1.0% propionatethioglycerol  0.1-0.5% monothioglycerol  0.1-0.5% norhydroguaianeticacid 0.01% ascorbic acid 0.015% esters thioglycolic acid 0.01-1.0%thiorthodi- 0.01-1.0% propionic acid

[0046] The preferred antioxidants are the metal ascorbates such as thealkali or alkaline earth metal ascrobates. Most preferably, the metalascorbate is sodium ascorbate (Aldrich Chemical Company, Inc.,Milwaukee, Wis. 53233.)

[0047] The pH of the formulation can be adjusted by the addition ofhydrochloric acid and/or sodium hydroxide. Typically the pH range of theproduct is from about 5.9 to about 8.5. The more preferred pH is about7.0.

[0048] Compositions of the invention can be supplied in unit dose vialsor ampoules, having the headspace filled by an inert gas. Preferably,the containers are protected from light and stored at temperatures fromabout 15° C. to about 30° C.

[0049] It is preferred that the aluminum content of the composition isless than or equal to one part per million (1 ppm). The aluminum contentcan be determined by atomic absorption spectophotometry and is typicallyreported in parts per billion (ppb). Preferably, the formulations of thepresent invention have less than about 1 ppm of aluminum during theshelf life of the product.

[0050] A preferred composition comprises 1.0 or 2.0 microgram (mcg) of1α, 25-dihydroxycholecalciferol, solubilizing agent, antioxidant,hydrochloric acid quantum sufficiat (q.s.), sodium hydroxide q.s., andwater for injection q.s. for one milliliter of solution (1 mL).

[0051] In a more preferred composition, each milliliter of solutionpreferably comprises 1.0 mcg of 1α, 25-dihydroxycholecalciferol, 4.0 mgof polysorbate 20, 2.5 mg of sodium ascorbate, hydrochloric acid q.s.,sodium hydroxide q.s., and water for injection q.s.

[0052] Polysorbate 20 is a nonionic surfactant which is commerciallyavailable as Tween® 20 (Polysorbate 20, Sigma Chemical Co., St. Louis,Mo., 63178).

[0053] Another preferred composition comprises 2.0 mcg of 1α,25-dihydroxycholecalciferol, 4.0 mg of polysorbate 20, 2.5 mg of sodiumascorbate, hydrochloric acid, q.s., sodium hydroxide q.s., and water forinjection q.s. Two examples of the preferred formulations of the presentinvention are presented below in Table 3. TABLE 3 Solution comprisingSolution comprising 1 microgram/milliliter 2 microgram/milliliter(mcg/ml) (mcg/ml) of 1α, 25-dihydroxy- of 1α, 25-dihydroxy-cholecalciferol cholecalciferol 1α, 25-dihydroxy- 2.0milligram/milliliter 4.0 milligrams/ cholecalciferol in (mg/ml)milliliter (mg/ml) polysorbate 20 concentrate, 575 mcg/g Polysorbate 202.0 mg/ml 0.0 mg/ml Sodium Ascorbate 2.5 mg/ml 2.5 mg/ml HydrochloricAcid q.s. q.s. Sodium Hydroxide q.s. q.s. Water for Injection q.s. q.s.

[0054] 3. Process for Making the Composition

[0055] Compositions of the present invention can be prepared by a novelprocess for controlling the total oxygen content in a pharmaceuticalcomposition. In general, an aqueous drug solution containing the desiredcomponents in suitable proportions is prepared and transferred in asuitable container via a process for establishing and controlling a lowoxygen environment.

[0056] In one aspect, the process for establishing and controlling a lowoxygen environment comprises the steps of:

[0057] a. flushing an empty container with an inert gas,

[0058] b. maintaining an inert gas environment by filling an aqueoussolution of 1α, 25-dihydroxycholecalciferol into said container whileconsistently flushing with an inert gas,

[0059] c. suitably minimizing the re-introduction of oxygen during steps(a) and (b) above and during transport of the container, and

[0060] d. sealing said container in a manner to minimize the presence ofoxygen in the container headspace.

[0061] The process enables the preparation of a stable, low aluminumcomposition of 1α, 25-dihydroxycholecalciferol in aqueous solution. Thecompositions have less than or equal to 2.0% oxygen in the headspace ofa container when determined immediately after the container sealing. Theoxygen content in the composition is determined volume by volumerelative to the total volume of the headspace.

[0062] One embodiment of the invention relates to the preparation of astable, low aluminum composition of 1α, 25-dihydroxycholecalciferolcomprising the steps of flushing an empty container in the presence ofan inert gas; maintaining an inert gas environment by filling an aqueoussolution of 1α, 25-dihydroxycholecalciferol into said container whileconsistently flushing with an inert gas; suitably minimizing there-introduction of oxygen during steps the previous steps and duringtransport of the container; placing an appropriate means of closure intothe opening of the filled container in a manner which allows theexchange of gas from an interior side of the container to an exteriorside of the container; vacuum treating the container headspace andpurging the container headspace with an inert gas; repeating the vacuumtreating and purging steps to control oxygen content in the containerheadspace; and sealing the container in a manner to minimize thepresence of oxygen in the container headspace. The preferred containerfor this embodiment is a vial having a suitable opening for flushingwith an inert gas and filling with a solution.

[0063] Another embodiment of the invention relates to the preparation ofa stable, low aluminum composition of 1α, 25-dihydroxycholecalciferolcomprising the steps of flushing an empty container with an inert gas;maintaining an inert gas environment by filling an aqueous solution of1α, 25-dihydroxycholecalciferol into said container while consistentlyflushing with an inert gas; suitably minimizing the re-introduction ofoxygen during the previous steps and during transport of the container;and flame-sealing the container in a manner to minimize the presence ofoxygen in the container headspace. The preferred container for thisembodiment is an ampoule.

[0064] In step (a), hereinafter the Preliminary Processing Stage (A), asuitable container for the composition of the invention is flushed withan inert gas. A means of conveyance transports the container to asuitable means for flushing the container with an inert gas. Theflushing means is connected to a source of inert gas, which is eitherindividually connected to the flushing means or is connected to aplurality of flushing means via a manifold to achieve a suitable flow ofgas. The inert gas source typically possesses a quality and puritysuitable for use in the manufacture of a pharmaceutical product. Theinert gas source can be attached by any suitable means that allowssupply of the gas into the flushing means. Preferably, the flushingmeans is a gassing needle that is connected in such a manner as to allowfor the flow of an inert gas into the container. The needle ispreferably attached by means consistent with industry practicesassociated with the maintenance of sterility, quality and purity of thedelivered inert gas, container and environment immediately surroundingthe container.

[0065] Introduction of the inert gas into an empty container displacespreviously existing gas contents of the interior of the containerthrough the opening of the container. The typical composition of thedisplaced gas mixture contains a combination of nitrogen and oxygen. Thepreferred inert gas for displacing the interior gas contents isnitrogen, which should be of a pharmaceutically acceptable grade.

[0066] In step (b), herein after the Filling Stage (B), the container isfilled with a desired solution through a suitable filling needle inconjunction with the introduction of inert gas. Preferably, the solutionis an aqueous pharmaceutical solution. To obtain a composition of theinvention, the drug solution comprises an aqueous solution of 1α,25-dihydroxycholecalciferol, a solubilizing agent, and an antioxidant aspreviously described.

[0067] It is preferred the container is contemporaneously filled withthe drug solution while purging with an inert gas. Nitrogen is thepreferred inert gas. The needle can be attached by any means suitablefor contemporaneously introducing a solution with an inert gas.Preferably, the needle is attached in a coaxial manner having a solutionfilling tube and a coaxial flushing tube. Flow parameters of the gas andthe solution can be adjusted to minimize the presence of oxygen or anyother undesirable gas in the headspace of the container.

[0068] To minimize the re-introduction of any undesirable gas in theprocess, the apparatus for carrying out the Preliminary PreparationStage (A) and the Filling Stage (B) of the process can be encased undera suitable shroud or enclosure to prevent infiltration of atmosphericgasses into the process environment. An interior volume enclosed by theshroud can be purged with an inert gas, which is preferably introducedvia an exterior manifold having outlets extending into the interiorvolume. The Preliminary Preparation Stage (A) and the Filling Stage (B)can be enclosed under the same shroud or, alternatively, a separateshroud can be fashioned for each stage. When each stage is encased in aseparate shroud or enclosure, the containers should be equally protectedfrom undesirable gas introduction, preferably, by means of an enclosureextension, a gassing tunnel or a shroud, which connects between theseparate enclosures surrounding the Preliminary Preparation Stage (A)and the Filling Stage (B). Preferably, the extension enclosure orgassing tunnel will be flushed with an inert gas delivered by anattached distribution manifold.

[0069] Outlets in the manifolds direct the flow of the inert gas insideof the shroud in a generally downward direction, displacing oxygen orother undesirable gasses within the interior of the shroud. The shroudis equipped with an exit opening, typically located at the bottom of theshroud, which allows displaced gasses to be purged from the interiorvolume of the chamber. Exemplary outlets in the manifold include holes,jets, needles, and the like or other similar orifices.

[0070] The inert gas from the manifold flows down past the container,displacing those gasses which have been purged from the interior of thecontainer during the Preliminary Preparation Stage (A) or the FillingStage (B). The inert gas, together with any other displaced gasses, willcontinue down well past and below the container, and exit the shroud orenclosure through a bottom opening. The shrouded environment allows forsuitable control over the gaseous environment encased within the shroudand provides an interior atmosphere that is extremely low in any contentof undesirable gasses, such as oxygen. It also affords an environmentwhich will minimize or reduce the potential for re-absorption of theoxygen or other undesirable gas into the desired solution previouslyintroduced into the container during Filling Stage (B).

[0071] Typically, the process then proceeds in one of two routes. In oneroute, the container is conveyed to a sealing station. In this route,the container is a suitable vessel having an opening that can bedirectly sealed by known methods in the art, for example, flame-sealing.A typical container of this kind is an ampoule. In another route, theopening of the container is fitted with a suitable closure, thecontainer and accompanying closure proceed into the sealed chamber forvacuum and purging, which are optionally repeated in succession, and thecontainers are fully sealed after completion of the vacuum and purgingprocess, preferably inside the sealed chamber. A suitable vessel havingan opening that can be sealed upon insertion of the appropriate means ofclosure is a vial. The vial is the preferred container, whichdemonstrates the preferred route.

[0072] In the preferred embodiment, vials are transported via the meansof conveyance and positioned in such a manner that an appropriate meansof closure may be inserted, hereinafter the Closure Stage (C).Preferably, the closure is a stopper which is selected in manner as tosuit the opening of the vial. A preferred stopper is a vented stopper,which allows the exchange of gas between the interior of the containerand the exterior environment of the container (which is coextensive withthe interior volume in the shroud). Such a stopper allows exchange ofthe undesirable gas in the headspace of the container to be displaced bythe preferred inert gas in the interior volume of the shroud. During theClosure Stage (C), inert gas is introduced by way of a manifold near thetop of the shroud to provide a downward flow of an inert gas, which canescape from an exit opening in the bottom of the shroud, as previouslydescribed for the Preliminary Preparation Stage (A) and Filling Stage(B).

[0073] The containers of either embodiment can be optionally accumulatedand collected to facilitate improved handling, hereinafter theAccumulation Stage (D). The containers are typically transferred to asuitable collecting means. Exemplary collecting means are trays, racks,and the like, or any other device suitable to hold a plurality ofcontainers. The containers can be transferred either automatically ormanually into the collecting means. The accumulation process issimilarly protected by a shroud or enclosure and flushed with an inertgas as previously described. The shroud is equipped with a suitableaccess door which can be used to remove the collected containers.Preferably, the access door is fitted with a means to seal any openingthat is created between the door and the shroud. Typically, thepreferred access door is fitted with a gasket or other similararrangement to provide a suitable seal.

[0074] Drawing a vacuum on the headspace of the container and purgingthe headspace for any remaining undesirable gas, hereinafter referred toas the Vacuum and Purging Stage (E), substantially removes any remainingundesirable gasses in process environment and in the composition. Asuitable apparatus for carrying out the vacuum and purging processconsists of a sealed chamber and one or more sealable access doors.Preferably, the chamber is equipped with hinged two access doors. Thechamber is fitted with a vacuum source and an inert gas source. Thevacuum source preferably comprises a motor, vacuum pump, vacuumregulator, and vacuum gauge, and is connect to the chamber via anexternal control valve. The inert gas source connects to the chamberalso via an independent external control valve. Containers are supportedby a suitable means for lifting the containers. The sealed chamber isalso suitably equipped with a means for regulating pressure within thechamber. The process can be regulated by any suitable means forcontrolling all aspects and steps of the process. Preferably, theprocess is controlled by a programmable logic controller (PLC). In thepreferred embodiment, the pressure transducer works in conjunction witha PLC to alternately draw a vacuum inside the chamber and flush thechamber with nitrogen.

[0075] The process is monitored to avoid evacuation vacuum levels thatasymptotically approach the critical vapor pressure or boiling point ofthe drug solution. In a typical process of the invention, the vacuumchamber controller or PLC program is streamlined to achieve vacuumlevels as close as is practicable to the boiling point of thecomposition. It is preferred that the environment within the processingchamber is allowed to reach equilibrium after the desired vacuum levelshave been achieved and then purged with inert gas to flush the entireinterior volume of the vacuum chamber. The single completion of thevacuum and purging steps results in a portion of the original oxygen andother undesirable gasses in the headspace being evacuated from thechamber.

[0076] Successive flushing and purging cycles further reduce totalconcentration of oxygen and other undesirable gasses in the headspaceand allow for controlled manipulation of the levels of undesirablegasses, particularly oxygen, in the container headspace. The methodresults in the compositions being exposed to extremely low levels ofoxygen and other undesirable gasses, which have not previously beenattained by known methods.

[0077] A composition of the invention is preferably treated under aseries of vacuum draws and oxygen purges performed within an environmentpurged of oxygen and other undesirable gasses. The vacuum treatmentdecreases the potential for re-absorption of oxygen into thecomposition, resulting in less dissolved or re-absorbed oxygen into thecomposition.

[0078] In the preferred embodiment, the vial containers can be suitablysealed while inside the sealed chamber flushed with inert gas by way ofa motive force device coupled to a shaft, which extends into theinterior of the vacuum processing chamber and is connected to asupporting platen. Preferably, a tray of containers is loaded onto thesupporting platen. The motive force device provides a suitable force tolift the platen toward an appropriate seal or sealing system to bringthe containers in position with a corresponding pre-positioned closure,such as a stopper. The pre-positioned closure is sealed into the openingof the container by compressing the container and its correspondingclosure. To ensure the integrity of the system, the appropriate seal orsealing system can be employed where the shaft passes through the wallof the processing chamber. The shaft retracts when closure of thecontainers is complete and the vacuum chamber controller or PLCactivates the pressure relief valve connected to the vacuum chamber toallow equalization of pressure inside the chamber. The containers can beremoved from the chamber and are suitable for further manufacturing, ifdesired.

[0079] Ampoules can be suitably sealed by flame-sealing, which is amethod well known to those in the art of pharmaceutical productmanufacturing.

[0080] The desired benefits of the described process are moreeffectively utilized in a composition of 1α, 25-dihydroxycholecalciferolin an aqueous solution, wherein the aqueous solution consistsessentially of a solubilizing agent and an antioxidant. The benefits aremost clearly demonstrated in a low aluminum formulation of 1α,25-dihydroxycholecalciferol prepared in an unit dose vial. However, itis believed the process of the invention can be used to control oxygencontent in nearly any pharmaceutical composition or unit doseformulation.

[0081] Schematic representations of the process and the apparatusemployed in the preferred embodiment of the process appear in FIGS. 1-8.Where a directional arrow is used in the schemata, it is intended thatthe arrowhead indicate the direction of flow of the inert gas. Theprocess of the invention and a suitable apparatus for carrying out theprocess can be better understood in context of the following moredetailed description of the drawings.

[0082] In FIG. 1, which refers to the Preliminary Processing Stage (A),a means of conveyance 1 transports a container 2 to a suitable gassingneedle 3. The gassing needle 3 is connected to a manifold 4 which isconnected to a suitable source of inert gas 5. Alternatively, eachgassing needle 3 is individually connected to the source of inert gas 5.The gas flows from the inert gas source 5 via the manifold 4 to thecontainer 2 to displace gas contents into the interior of the container.

[0083] An independent source of inert gas 6 enters the environmentexterior to the container via a manifold 7. A shroud 8 encloses aninterior volume 9. The interior volume 9 is filled with an inert gas 6delivered by the manifold 7.

[0084] As illustrated in FIG. 2, which refers to the Filling Stage (B),container 2 is filled with solution via a coaxial filling needle 10. Thefilling needle 10 is used in conjunction with manifold 11 to supply asource of inert gas 12 while introducing a desired solution 14. Theinert gas 12 flows into container 2 via the manifold 11 to purge the gascontents in the interior of the container to the exterior of thecontainer. The apparatus is contained under shroud 8 and the interiorvolume 9 can be filled with an inert gas. Alternatively, the apparatusis contained under a separate shroud. Gas source 15 flows manifold 15 bto purge the interior volume 9 with an inert gas.

[0085] In FIG. 3, the gassing needle 3 feeds the inert gas 5 intocontainer 2. The inert gas flows in the direction of the arrows from thegassing needle 3 into the container 2 and is displaced into the exteriorenvironment of container 2.

[0086] According to FIG. 4, the preferred means for attaching thefilling needle 10 is by using a coaxial flushing means which comprises asolution filling tube 16 and a coaxial flushing tube 17. Inert gas 12flows into the headspace of container 2 via the coaxial flushing tube17, while the desired solution 14 is introduced via filling tube 16.

[0087] In FIG. 5, which refers to the Closure Stage (C), gassingmanifold 18 feeds the inert gas onto and around the container whileoutside of shroud 8. The means of conveyance 1 brings container 2 to aposition that is suitable for receiving via a placing mechanism 21 anappropriate means of closure 22. The means of closure 22 is placed onthe container 2 in a manner that will allow the exchange of gas betweenthe interior of the container and the atmosphere outside of thecontainer. The interior environment 9 is filled with an inert gas 19 bymeans of a suitable manifold 20. The container 2 is conveyed by means 1to the exterior of the shroud 8. The external environment is protectedby a shroud or covered tunnel 25, and purged with an inert gas 23 whichis distributed by a manifold means 24.

[0088] As described in FIG. 6, which refers to the Accumulation Stage(D), the container 2 is conveyed by means 1 into the tunnel enclosure25. A transfer device 26 moves the container 2 in an environment purgedby inert gas 27, which is delivered via manifold 28. The container 2 istransferred into a collecting means 29 that can be removed via an accessdoor 30 located at a suitable position in shroud 8. Gasket 31 seals theaccess door 30 to the shroud.

[0089] As illustrated in FIG. 7, which refers to the Vacuum and PurgingStage (E), sealed chamber 32 is equipped with a gasketed access door 33connected by hinge 34 and supplemented by gasket 35. A tray ofcontainers is carried by supporting platen 36. Control valves 37 and 38operate to allow inflow and outflow of the gas and vacuum, respectively,and are attached to the sealed chamber. Vacuum source 39 is attached andcontrolled by valve 38. An inert gas source 40 is attached to ancontrolled by valve 37. Shaft 41 extends to the interior of the vacuumchamber and attaches on one end to supporting platen 36. Motive forcedevice 42 couples to the other end of shaft 41 and provides air orhydraulic force to control the movement of supporting platen 36. Anappropriate seal or sealing system 43 closes any gap between the shaft41 and the chamber 32 where the shaft enters the chamber. A pressuretransducer 44 is suitably connected to the chamber 32.

[0090] According to FIG. 8, the inert gas source 40 comprises a gasstorage vessel 45, pressure regulator 46 and pressure gauge 47. Thechamber 32 is equipped with a pressure release valve 48. A stored sourceof energy 49 is suitably connected to the motive force device 42 toprovide air or hydraulic energy, which can be controlled via valve 50. Ameans for controlling all aspects and steps of the process 51, such as aprogrammable logic controller (PLC), connects to the apparatus viacontrol valves 37, 38, 50, and a pressure controller/display mechanism52, which is associated with the pressure transducer 44. Vacuum source39 comprises motor 53, vacuum pump 54, vacuum regulator 55, and vacuumgauge 56.

[0091] 4. Sterilization of the Composition

[0092] Formulations of the invention can be aseptically filled orterminally sterilized by a variety of sterilization techniques.Exemplary sterilization techniques useful for preparing the compositionsinclude, but are not limited to, autoclaving, gamma radiation, andelectron beam sterilization techniques. Preferably, the formulations ofthe invention are terminally sterilized. The preferred technique forterminally sterilizing the formulations is autoclaving.

[0093] Suitable conditions for autoclaving the formulations include butare not limited to, terminal sterilization at F_(o) of 8 to F_(o) of 16,which denotes a sterilization cycle run at 121.11° C. for 8 to 16minutes, respectively, with saturated steam.

[0094] 5. Stability of the Compositions

[0095] The compositions of the present invention provide novelformulations with low levels of oxygen in the solution, whichdemonstrate improved stability in color and potency of the 1α,25-dihydroxycholecalciferol active agent.

[0096] Generally, terminally sterilized products are prepared inaccordance with color standards in accordance with the 1995 U.S.Pharmacopiea/National Formulary, Edition No. 23, pages 1779-1780 and1860-1861. A composition of the invention will typically exhibit a colorvalue of less than about 100 APHA units when measured immediately aftercontainer sealing. A preferred composition has a color value of lessthan about 400 APHA units when stored for three months at a temperatureof about 40° C.

[0097] Potency levels of the 1α, 25-dihydroxycholecalciferol activeagent typically measure from about 90% to about 120% of the formulationduring the shelf life of the product. Typically the product shelf lifeis from about 12 months to about 18 months. Compositions, unit dosesystems, and products made by the claimed process can be prepared tohave the potency and color values as described above.

[0098] The following Example is provided to illustrate the formulationsof the invention and is not intended to limit the scope of the inventionin any way.

EXAMPLE 1

[0099] A calcitriol aqueous solution with the following composition wasprepared: TABLE 2 Ingredient Amount per ml Calcitriol 2 mcg Polysorbate20 4 mg Sodium 2.5 mg ascorbate Water q.s.

[0100] The formulation was prepared as follows:

[0101] 1α, 25-dihydroxycholecalciferol (5.75 mg) was accurately weighedin a nitrogen purged glove box and quantitatively transferred into 100 gof polysorbate which was preheated to 65° C. The nitrogen purge wascontinued in the glove box while mixing, until all the 1α,25-dihydroxycholecalciferol powder was dissolved in polysorbate. The 1α,25-dihydroxycholecalciferol concentrate was kept at 5° C. with nitrogenheadspace protection and light protection.

[0102] To 900 ml of water for injection, was added and dissolved 2.5 mgof sodium ascorbate while maintaining nitrogen sparge in the solution.The nitrogen sparge was switched to nitrogen blanket and maintained forthe entire process. Then 2.0 mg of polysorbate was added with mixing.Accurately weighed 2.0 g of 1α, 25-dihydroxycholecalciferol concentratein polysorbate, 575 mcg/g was added into the solution with gentlemixing. The solution was added quantum sufficiat (q.s.) with water forinjection to obtain a 1 L final volume. The solution was mixed gentlyuntil uniform. The pH of the solution was adjusted to 8.3-8.7 withaddition of either hydrochloric acid or sodium hydroxide.

[0103] The solution was filled into amber vials with nominal fill of1.25 ml per vial via the process previously described in thespecification. The oxygen content in the headspace of the containermeasured immediately after sealing the container was 0.4% by GC(Shimadzu, model GC-17A). After sealing, the vials were terminalsterilized. The pH of the composition was about 7.0. Calcitriol potency,color and aluminum values of the sample were tested at initial, 3 monthsstored inverted at 40° C. and 9 months stored inverted at 25° C. Thepotency was tested by using HPLC (Hewlett Packard model, HPLC 1100), thecolor data were generated on a Gardener calorimeter (BYK Gardener ColorSpectrophotometer) and a graphite furnace atomic absorption spectrometer(Perkin Elmer model 4110ZL) was used in the aluminum testing utilizingmethodologies known or readily developed by those skilled in the art.The results are presented in Table 4. TABLE 4 Time of testing Potency %Aluminum, ppb Color, APHA unit Initial 111  82  41 3M, 40° C., inverted105 218 193 9M, 25° C., inverted 108 — 278

[0104] The samples showed a stable aqueous formulation for calcitriolthat does not contain a buffer and a chelating agent. The formulationwas sterilized by autoclave and contained only a trace amount ofaluminum in the sterile solution.

EXAMPLE 2

[0105] A calcitriol solution with the same composition as in Example 1was prepared and filled into vials with nominal fill 1.25 ml. Theheadspace oxygen content in the vials was controlled at different levelsby the process described in this invention. The headspace oxygen contentwas measured immediately after sealing the vials. Calcitriol potency andcolor were measured at initial and 3 months stored at 40° C. The testdata are summarized in Table 5. TABLE 5 Color, Potency (%) Color, APHAunits Potency (%) 3 months at APHA units 3 months Headspace Initial 40°C. Initial at 40° C. 0.2% Oxygen 115 111 11 234 0.5% Oxygen 117 110 27269 1% Oxygen 115 109 24 235 1.5% Oxygen 116 110 27 364 2% Oxygen 116109 39 401 4% Oxygen 117 104 37 495

[0106] The data in Table 5 demonstrated that calcitriol potency remainsstable for the sample vials with headspace oxygen levels less than orequal to 2%. Lower oxygen levels in the headspace correlated with thelower color value developed during storage. At 40° C., 3 months, thecolor value for the 4% headspace oxygen sample is almost twice as highas that in samples with less than 1% headspace oxygen.

What is claimed is:
 1. A stable composition comprising a therapeuticallyeffective amount of 1α, 25-dihydroxycholecalciferol in an aqueoussolution having less than or equal to 2.0% oxygen in the headspace of acontainer when determined immediately alter container sealing, saidsolution consisting essentially of a solubilizing agent and anantioxidant.
 2. The stable composition according to claim 1, wherein thecomposition measures from about 90% to about 120% potency whendetermined during the shelf life of the composition.
 3. The stablecomposition according to claim 1, wherein the composition measures amean color value of less than about 150 APHA units immediately aftercontainer sealing.
 4. The stable composition according to claim 1,wherein the composition measures a mean color value of less than about400 APHA units when determined after three months of storage at atemperature of about 40° C.
 5. The stable composition according to claim1, wherein the pH is from about 5.9 to about 8.5.
 6. The stablecomposition according to claim 5, having an aluminum content of lessthan or equal to about one part per million (1 ppm) during the shelflife of the composition.
 7. The stable composition according to claim 6,wherein the composition is terminally sterilized.
 8. The stablecomposition according to claim 1, wherein the solubilizing agent isselected from dimethylacetamide, polyethylene glycol 400 (PEG 400),polyethylene glyclol 200 (PEG 200), ethanol, isopropanol,1,3-butanediol, propylene glycol, dimethylsulfoxide, glycerin,polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate
 80. 9.The stable composition according to claim 8, wherein the solubilizingagent is polysorbate
 20. 10. The stable composition according to claim9, wherein the solubilizing agent comprises at least 0.32% weight byweight (wt./wt) polysorbate
 20. 11. The stable composition according toclaim 1, wherein the antioxidant is selected from ascorbic acid, metalascorbates, sodium bisulfite, sodium metabisulfite, ascorbyl palmitate,sodium sulfite, sodium formaldehyde sulfoxylate, acetone sodiumbisulfite, tocopherol, thioglycerol, monothioglycerol,norhydroguaianetic acid, ascorbic acid esters, thioglycolic acid,thiorthodipropionic acid, and dilaurylthiodipropionate.
 12. The stablecomposition according to claim 11, wherein the antioxidant comprisesfrom about 0.001% wt./wt to about 1.5% wt./wt of the aqueous solution.13. The stable composition according to claim 11, wherein theantioxidant is ascorbic acid or metal ascorbate.
 14. The stablecomposition according to claim 13, wherein the antioxidant comprises0.2% wt./wt. to 1.0% wt./wt. sodium ascorbate.
 15. The stablecomposition according to claim 1, having 1.0 to 2.0 microgram (mcg) of1α, 25-dihydroxycholecalciferol, solubilizing agent; antioxidant;hydrochloric acid quantum sufficiat (q.s.), sodium hydroxide q.s., andwater for injection q.s. for 1 milliliter (mL) of aqueous solution. 16.The stable composition according to claim 15, having 1.0 mcg of 1α,25-dihydroxycholecalciferol, 4.0 mg of polysorbate 20, 2.5 mg of sodiumascorbate, hydrochloric acid q.s., sodium hydroxide q.s., and water forinjection q.s. for 1 mL of aqueous solution.
 17. The stable compositionaccording to claim 16, wherein the pH is about 7.0.
 18. The stablecomposition according to claim 1, having 2.0 mcg of 1α,25-dihydroxycholecalciferol, 4.0 mg of polysorbate 20, 2.5 mg of sodiumascorbate, hydrochloric acid q.s., sodium hydroxide q.s., and water forinjection q.s. for 1 mL of aqueous solution.
 19. The stable compositionaccording to claim 18, wherein the pH is about 7.0.
 20. A stablecomposition comprising a therapeutically effective amount of 1α,25-dihydroxycholecalciferol in an aqueous solution having less than orequal to 2.0% oxygen in the headspace of a container when determinedimmediately after container sealing, said solution consistingessentially of a solubilizing agent and an antioxidant, said solutionhaving an aluminum content of less than or equal to about one part permillion (1 ppm) during the shelf life of the composition.
 21. The stablecomposition according to claim 20, wherein the composition measures fromabout 90% to about 120% potency during the shelf life of thecomposition.
 22. The stable composition according to claim 20, whereinthe composition measures a mean color value of less than about 150 APHAunits immediately after container sealing.
 23. The stable compositionaccording to claim 20, wherein the composition measures a mean colorvalue of less than about 400 APHA units when stored for three months ata temperature of about 40° C.
 24. The stable composition according toclaim 20, wherein the pH is from about 5.9 to about 8.5.
 25. The stablecomposition according to claim 20, having aluminum content of less thanor equal to about 1 ppm of the solution during the shelf life of thecomposition.
 26. The stable composition according to claim 20, whereinthe composition is terminally sterilized.
 27. A unit dose systemcomprising an aqueous solution of a therapeutically effective amount of1α, 25-dihydroxycholecalciferol in a sealed vessel having less than orequal to 2.0% oxygen in the headspace of a container when determinedimmediately after container sealing, said solution consistingessentially of a solubilizing agent and an antioxidant.
 28. The unitdose system according to claim 27, wherein the aqueous solution isessentially free of buffer or chelating agent.
 29. The unit dose systemaccording to claim 27, having less than about 1 ppm of aluminum duringthe shelf life of the composition.
 30. The unit dose system according toclaim 29, wherein the unit dose system is terminally sterilized.
 31. Aprocess for preparing a stable, low aluminum composition of 1α,25-dihydroxycholecalciferol, comprising the steps of: a. flushing anempty container with an inert gas, b. maintaining an inert gasenvironment by filling an aqueous solution of 1α,25-dihydroxycholecalciferol into said container while consistentlyflushing with an inert gas, c. suitably minimizing the re-introductionof oxygen during steps (a) and (b) above and during transport of thecontainer, and d. sealing said container in a manner to minimize thepresence of oxygen in the container headspace.
 32. The process accordingto claim 31, further comprising the steps of: a. placing an appropriatemeans of closure into the opening of the filled container obtained instep (c) of claim 31 in a manner which allows the exchange of gas froman interior side of the container to an exterior side of the container;b. vacuum treating the container headspace having an appropriate meansof closure and purging the container headspace with an inert gas, c.repeating the vacuum treating and purging step of (b) above to controlthe oxygen content in the container headspace before suitably sealingsaid container.
 33. The process according to claim 32, wherein thecontainer is a vial.
 34. The process according to claim 31, wherein thecontainer is an ampoule.
 35. The process according to claim 31, whereinthe container is flame-sealed.
 36. The product prepared by the processaccording to claims 31, 32, 33, 34 or
 35. 37. The product prepared bythe process according to claim 31 or 32, comprising a therapeuticallyeffective amount of 1α, 25-dihydroxycholecalciferol in an aqueoussolution having less than or equal to 2.0% oxygen in the headspace of acontainer when determined immediately after container sealing, saidsolution consisting essentially of a solubilizing agent and anantioxidant.
 38. The product prepared by the process according to claim31 or 32, comprising a therapeutically effective amount of 1α,25-dihydroxycholecalciferol in an aqueous solution having less than orequal to 2.0% oxygen in the headspace of a container when determinedimmediately alter container sealing, said solution consistingessentially of a solubilizing agent and an antioxidant, and having analuminum content of less than about 1 ppm during the shelf life of thecomposition.
 39. A unit dose system comprising an aqueous solution of1α, 25-dihydroxycholecalciferol in a sealed vessel, wherein each mL ofthe aqueous solution consists essentially of: a. about 1.0 mcg to about2.0 mcg of 1α, 25-dihydroxycholecalciferol, b. 4.0 mg of polysorbate 20;c. 2.5 mg of sodium ascorbate; d. hydrochloric acid q.s.; e. sodiumhydroxide q.s.; and f. water for injection q.s.; said unit dose systemhaving less than or equal to 2.0% of oxygen in the headspace.